View Abstract

A7604 - Single-Cell RNA-seq Highlights Fibroblast Heterogeneity Among Models of Tissue Fibrosis
Author Block: K. Sun1, T. Tsukui1, Y. Chang2, I. Driver3, D. Sheppard1; 1Medicine, UCSF, San Francisco, CA, United States, 2Medicine, University of California, Irvine, Orange, CA, United States, 3UCSF, San Francisco, CA, United States.
RATIONALE
Fibrosis is a pathological sequela of tissue injury, and is characterized by excessive accumulation of fibrillar collagens (especially collagen I and III) and other secreted ECM proteins. The presumption of current paradigm has been that there is a relatively homogenous population of αSMA-expressing myofibroblasts responsible for tissue fibrosis; however, accumulating evidence suggests that this paradigm is overly simplistic. To address this critical question, we purified collagen-expressing cells from multiple models of tissue fibrosis in Col1a1-GFP mice, followed by transcriptome analysis via Single-cell RNA-seq.
METHODS
Cells were purified from Col1a1-GFP mice with and without induction of bleomycin-induced pulmonary fibrosis, CCl4-induced hepatic fibrosis, unilateral ureteral obstruction-induced renal fibrosis, or bleomycin-induced skin fibrosis, followed by Single-cell RNA-seq, transcriptome analysis and target validation.
RESULTS
Based on unsupervised principal components analysis (PCA) compressing 13 statistically significant PCs into 2-dimensional t-SNE plot, we found multiple distinct clusters of Col1a1-GFP+ cells from 4 fibrosis models we used, which highlights the heterogeneity among these models. Repeating PCA focusing on Col1a1-GFP+ cells from untreated and bleomycin-treated lungs, 5 distinct populations were identified: 4 clusters contained cells from bleomycin-treated lungs, and 1 cluster contained cells from both. We then conducted functional analysis of these 5 clusters in lungs via Database for Annotation, Visualization and Integrated Discovery. From bleomycin-treated lungs, we identified two novel clusters expressing fibrotic genes, one cluster with characteristics of myeloid cells, and one cluster with characteristics of epithelial cells. The cluster containing cells from untreated and bleomycin-treated lungs was annotated as lipofibroblasts. To further validate the results, our flow cytometry showed increasing of Col1a1-GFP+CD45+ cells (myeloid cluster) in bleomycin-treated lungs in comparison to untreated lungs. In addition, we performed Immunohistochemistry on bleomycin-treated lungs from Col1a1-GFP;αSMA-RFP dual reporter mice, and we found co-localization of Col1a1-driven GFP with several cluster markers, such as Sftpd for “Epithelial Cluster” and CD45 for “Myeloid Cluster”. Interestingly, only some of these cells co-expressed αSMA, suggesting that αSMA is not a completely specific marker of collagen-producing cells upon injury. Finally, we co-stained pro-collagen and several these markers in human IPF lungs, which supports the existence of cells with identified characteristics.
CONCLUSIONS
Our data highlight the substantial fibroblast heterogeneity among models of tissue fibrosis. We also validated the existence of collagen-expressing cells with different specific markers in vivo and in patients’ tissue. Better understanding of these cells is crucial to design novel therapeutic targets for fibrotic diseases.